Thymidine

Dynamics of Replication and Nuclear Localization of the B Chromosome in Kidney Tissue Cells in Astyanax scabripinnis (Teleostei: Characidae)

Zelinda Schemczssen-Graeff,1 Patr´ıcia Barbosa,2 Jonathan Pena Castro,2 Maelin da Silva,1 Mara Cristina de Almeida,1 Orlando Moreira-Filho,1,2 and Roberto Ferreira Artoni1,2

Abstract

B chromosomes are extra genomic compounds found in different taxonomic groups, including plants and animals. Obtaining patterns of resolutive chromosomal bands is necessary to understand the nuclear organization, variability and nature of B chromosome chromatin and possible transcriptional regions. In this study, we analyzed 35 Astyanax scabripinnis specimens sampled from Fazenda Lavrinha, a stream in the Paraı´ba do Sul river basin, Brazil. Through the incorporation of the thymidine analog 5¢-bromo-2¢-deoxyuridine (5-BrdU) in vivo, it was possible to recognize the replicating regions of the B chromosome at the beginning of the S phase, differentially characterized in relationship to the regions of late replication. In this perspective, it is possible to suggest that the B chromosome of this species possesses a territory and the chromatin accessible for transcription, especially in the light (i.e., early replicating) bands (p1.1; p1.3; and p2.1 and q1.1, q1.3, q2.1, and q2.2). The latereplicating regions are corresponding to the blocks of constitutive heterochromatin. They show a preferential accumulation of satellite DNA As51. By the use of the fluorochrome chromomycin A3 (CMA3), it was possible to identify GC-rich chromosomal regions, corresponding to late-replicating parts of genome, confirming the revealed data by the replication banding and C-banding. In addition, the analysis by confocal microscopy in kidney cells indicates the location of a peripheral anchorage of this chromosome in the nuclear lamina, reinforcing the idea of downregulation of the associated regions.

Keywords: DNA replication, 3D FISH, chromosome banding, chromosome territories, confocal microscopy

Introduction

In A. scabripinnis, the origin of the B macrochromosomes was initially attributed to events of meiotic nondisjunction chromosomesare extra genomic compounds occurring based on the comparative inferences from the size and morin different taxonomic groups, including plants and an- phology of these B chromosomes when compared to the first imals, Since their discovery in the beginning of the 20th century by Wilson,3 B chromosomes in organisms have been extensively investigated.4–7 Recent studies have focused on the evolution and biology of B chromosomes, highlighting the occurrence of genes on these extra genomic elements (revisited in Houben et al.2).
In Neotropical fishes, 4% of the more than 1000 species with known karyotypes have B chromosomes.8,9 From these, seven species belong to the genus Astyanax, namely10 A. fasciatus, A. schubarti,11 A. altiparanae,12 A. bockmanni,13 A. goyacensis,14 A. paranae,15 and A. scabripinnis.10,16 pair of standard A chromosomes.
Recently, the more accepted theory assumes that the B chromosome of A. scabripinnis had originated through an emergence of isochromosomes from the acrocentric pair 24.17 This hypothesis is based on the presence of an interstitial heterochromatic band in the long arm of this chromosome pair, displaying an equilocal position on both arms of the B chromosome, in addition to a strongly marked band on the two arms of the B chromosome.17 This evidence is corroborated by the chromosomal location, of As51 satellite DNA after fluorescence in situ hybridization (FISH),18 and the chromosome pairing during meiosis.19
Populations of A. scabripinnis from the Atlantic forest on the Brazilian coast (Campos do Jorda˜o, Sa˜o Paulo) have attracted special attention in evolutionary studies, considering that this region is *2000m in altitude and the populations live isolated at the headwaters of small streams,16 many of them with the presence of the B chromosome (revisited by Moreira-Filho et al.20).
One ofthe central problemsof the comparative cytogenetics in fishes is the difficulty to obtain resolute patterns of longitudinal chromosomal bands.21 An alternative is to obtain the replication bands by the incorporation of the base analogs during cellular division,22 which is based on the incorporation of the thymidine analog 5¢-bromo-2¢-deoxyuridine (5-BrdU). It can be a strategy to delimit the regions with a possible transcriptional activity on B chromosomes.
Using the replication banding method, Maistro et al.23 demonstrated that the B chromosome of A. scabripinnis possess a late-replicating pattern due principally to its heterochromatic nature.10,17 Nonetheless, the replication timing in the whole chromosome and intercalary regions still unclear. In addition, the nature of the B-chromosome chromatin is an unresolved question, despite the mapping of microsatellite sequences.24
The chromosomes occupy separated territories in the interphase nucleus of eukaryotic cells.25 A co-regulation model proposed that more centralized regions in the nucleus are enrichment with transcriptionally active genes while more periphery regions content inactive regions of the genome.26
On the other hand, new models of genetic regulation within the interphase nucleus have been proposed. The definition of maps with high resolution of the chromatin interaction allows us to identify the so-called topologically associated domains (TADs), which are based and identified according to the expression activity, replication timing, or the epigenetic landscape proteins associated with them. In addition, other chromosome domains can be defined by the interaction between neighboring regions, such as laminaassociated domains (LADs), defined by chromosomal interactions with a nuclear lamina and their a correlation with low gene expression and late replication timing. Thus, such mechanisms have been understood and identified with respect to cell type-specific gene expression.27 Genome-wide data from different organisms are increasingly available. The interpretation of this information against 3D conformations of the chromosomes in the nucleus and their territories, with an effect on the biology of organisms, will be the biggest challenge in the coming years. On the other hand, the nuclear territory of B chromosomes, especially in fishes, is still completely lacking.
Thus, this study aims to investigate whether the B chromosome of A. scabripinnis possesses transcriptional regions, using the replication pattern, the chromatin nature, and their distribution in the interphase nucleus of kidney cells.

Materials and Methods

Biologic samples

We used 35 A. scabripinnis specimens from Fazenda Lavrinha,a streaminthe Paraı´badoSul river basin(S2243¢09,6†/ WO 4525¢38,5†). The specimens were identified and received a voucher number from Nu´cleo de Pesquisas em Limnologia, Ictiologia e Aquicultura of Universidade Estadual de Maringa´ (NUP no. 17482). This research had the authorization from the ethics committee of the State University of Ponta Grossa (Protocol N 04509/08) and license to collect biological material by Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renova´veis (IBAMA) under n15115-1.

Chromosome preparations

Mitotic chromosomes were obtained from the kidney cells by direct preparation described in Bertollo et al.28 and shortterm culture according to Fenocchio et al.29 The incorporation of thymidine analog 5-BrdU and the revelation of bands followed the established protocol of Giles et al.30 The constitutive heterochromatin was performed by the C-banding method.31 For staining with the base-specific fluorochromes chromomycin A3 (CMA3) and 4¢,6-diamidino-2phenylindole (DAPI), the protocol described by Schweizer32 was followed.

Preparation of cells for confocal microscopy

Part of the kidney cell suspension was resuspended in fetal bovine serum (Sigma-Aldrich Brasil Ltda) and dripped onto a previously polysin-coated slide (10mg/mL), and left in the oven at 37C for 1h. The cells were drained of excess fetal bovine serum and washed in 0.3·phosphate-buffered saline buffer for 40s. Subsequently, the fixation in 4% w/v paraformaldehyde took place. After this procedure, the slides could be used for FISH procedures. The 3D images were captured using a Leica TCS SP8 laser scanning confocal microscope with LAS AF SP8 3D visualization software, with 2lm capture cut.

Fluorescent in situ hybridization

The As51 satellite DNA described by Mestriner et al.18 was obtained from the nuclear DNA of A. scabripinnis using the primers Fw 5¢-GGTCAAAAAGTCGAAAAA-3¢ and Rv 5¢-GTACCAATGGTAGACCAA-3¢ in 35 cycles in the Eppendorf Mastercycler 1min at 95C, 45s at 56C, 1min at 72C, and 5min at 72C. The As51 satellite DNA probe was marked by the nick translation using biotin 14-dATP (Bionick Labeling System; Invitrogen-Thermo Fisher Scientific, SP, Brazil) in dry bath Loccus Biotecnologia TM (1h and 30min at 15C after 15min at 65C).
FISH was performed using the protocol of Pinkel et al.33 with modifications according to Vicari et al.34 The protocol has been adapted for hybridization in high stringency conditions (2.5ng/mL probe, 50% deionized formamide, 10% dextran sulfate, and 2·buffer saline sodium citrate [SSC] at 37C overnight). After the hybridization, the slides were washed in a 50% formamide/0.2·SSC solution at 42C for 20min and 4·SSC/0.05% Tween at room temperature for 10min. The signal detection was performed with Streptavidin conjugated with Alexa Fluor 488 (Thermo Fisher Scientific). The chromosomes were counterstained with DAPI (0.2lg/mL) diluted in antifade solution (Fluka).
Chromosomes and nuclei were analyzed under an epifluorescence microscopy Zeiss AxioCam MRm with software ZEN pro 2011 (Carl Zeiss) and confocal microscopy Leica TCS SP8 with software LAS AF SP8 3D visualization. The best metaphases were photographed and the karyotype was organized in Photoshop, version CC 2015. The diagram was assembled with the Corel Draw v. X7.

Results

The conventional analysis of the karyotype of A. scabripinnis evidenced a karyotype macrostructure with a presence of one polymorphic metacentric B chromosome in the population. These data validate previous studies with specimens from the population of the Fazenda Lavrinha stream with 2n=51 chromosomes (6m + 22sm +10st +12a + 1Bm), without an occurrence of morphologically differentiated sex chromosomes.35
The cells treated with 5-BrdU showed a banding pattern corresponding with a heterogeneous replication timing on the chromosomes of A. scabripinnis (Fig. 1). The B chromosome possessed a longitudinal banding pattern compatible with the early replication (bright bands), replicating (p1.3; p1.1; p2.2 e q1.3; q1.1; q2.3; q2.1), and also with late replication (p2.1; p1.2 e q1.2; q2.4; q2.2) (Fig. 2).
The late-replicating regions were coincident with the blocks of constitutiveheterochromatinand preferentiallocalizationof repetitive sequences As51 satellite DNA (Fig. 2). The fluorochrome CMA3 allowed us to visualize CG-rich regions with high contrast, coincident with late-replicating regions (Fig. 2). The C-banding method showed heterochromatic region patterns similar to previous studies in Astyanax species.10,17
The utilization of a confocal laser scanning microscopy associated the FISH-based chromosomal mapping of As51 satDNA (Fig. 3a, b), identified that this sequence occupies a peripheral domain in the interphase nucleus of kidney cells (Fig. 3c).

Discussion

In this study, we obtained a replication banding pattern through in vivo treatment with 5-BrdU (6–7h), which enabled more precise identification of homologs into 25 pairs and yielded multiple bands on the B chromosome. Previous studies revealed replication bands in A. paranae,23 A. altiparanae, A. schubarti, A. fasciatus, and A. scabripinnis.36 However, in obtaining these markers by 5-BrdU, with intention of use for interspecific karyotype comparison, this marker fell into disuse in fishes, due to a lack of comparative patterns of these bands in identifying chromosomal homologies.22 Thus, new studies of B chromosomes using 5-BrdU in some of these species have ceased, due to the dynamic nature of the band replication obtained with this methodology. We return to the subject in this work, performing an integrative analysis of the structure, replication dynamics, and potential functional regions of these supernumerary chromosomes.
In this study, we find regions corresponding to light and dark replication bands on the B chromosome of A. scabripinnis. The obtained pattern shows that this chromosome, although generally appearing completely heterochromatic, has a heterogeneous nature with regard to the chromatin composition. In addition, it can be inferred that the replication of this chromosome occurs at different stages of the S phase of the cell cycle.
AT-rich regions are relatively underrepresented in opposite to GC-rich gene.37 With the fluorochrome CMA3, it was possible to evidence regions rich in CG, and CMA3+ regions poor in genes, confirming what was identified by the incorporation of 5-BrdU and C-banding. Cristiano et al.38 found the same pattern for CMA3 in Melitoma segmentaria. Other bees have the same cytogenetic divergence as Melipona in relationship to heterochromatin content and have unique patterns of CMA3 accumulation, showing that heterochromatin is heterogeneous in relationship to its composition, with some blocks rich in AT—and others in CG pairs.39
Our results indicate that the B chromosome of A. scabripinnis has a chromosomal compartmentalization, reminiscent of isochores observed in mammals (Fig. 2), which are involved in genome compartmentalization and support the tissue-specific transcriptional regulation in all metazoa.40,41 In the yeast, Saccharomyces cerevisiae, some chromosomal regions replicate at different times,42,43 as well as in mammals and other eukaryotes.37
In this perspective, it is possible to suggest that the B chromosome of A. scabripinnis studied, herein, exhibits chromatin accessible for transcription, particularly in the light bands corresponding with early replication. Regardless to their possible origin, the chromosomal fragments that include gene sequences are potential components of any B chromosome and because there is a relaxed selective pressure over the genes and others sequence on the B chromosomes, evolutionarily newly formed B chromosomes must be richer in functional gene sequences than the evolutionarily older ones.39,44
The epigenetic marks 5-methylcytosine and 5-hydroxymethylcytosine in the B chromosome of Astatotilapia latifasciata, for example, may suggest it transcriptionally active status. There is a correlation with the B chromosome pres- replicating regions were expected due to the heterochromatic ence and epigenetic effects.45 nature of the B chromosome. Regions on the B chromosome
The regions marked by FISH with a probe corresponding marked by As51-specific probe are coincident with methylto As51 satDNA coincided with the late-replicating hetero- ated sequences and accumulations of specific transposable chromatic regions on A chromosomes and it especially elements (LINE and Tc1-Mariner), suggesting that these highlighted such regions on the B chromosome. Late- regions are being silenced in the genome (Barbosa et al.,unpublished data). In addition, these regions are involved in the anchorage of this chromosome to the nuclear lamina, shown by confocal microscopy (Fig. 3c).
The sites where the chromosomes are anchored within the nucleus and where they form domains correspond directly to the association of chromatin with the nuclear lamina, thus forming LADs.25 These domains are involved in the downregulation of gene expression. Actively transcribed genes are usually found clustered in the center of interphase nucleus, while the inactive parts of the genome reside more toward the periphery of the nuclei.46 The repositioning hypothesis for the B chromosome of A. scabripinnis should be tested, given the heterogeneity of replication bands obtained here for this chromosome.
Hypomethylated centromeric regions of the corn B chromosome, for instance, become hypermethylated when this chromosome is transferred to oats.47 It is very likely that the regions corresponding to the early replicating pericentromeric bands (p1.1 and p1.3, hypomethylated and unlabelled by 5-methylcytosine according to Barbosa et al. unpublished data) may harbor genes related to centromeric function and maintenance of B chromosomes. Some studies have reported the presence of genes related to cell division in B chromosomes,48 to microtubules, structure of the kinetochore, and recombination in fishes.49
Moreira-Filho et al.20 reviewed the known information for B chromosome in A. scabripinnis, their presence, origin, frequency, and distribution in natural populations, and concluded that these approaches need further complementary studies, so that the presence and significance of B chromosomes for the species are better understood. One of the questions that still remains unresolved deals with the possible effects of the B chromosomes on their carriers. The mapping of euchromatic regions onthe BchromosomeofA.scabripinnisobtainedhere, aswellastheir locationininterphasenuclei, isfundamental for the mapping of gene sequences and studies of the biological function of these enigmatic extra chromosomes.

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